The long-term goal of this research project is to understand the molecular mechanism of force production in muscle through 3-D visualization of crossbridge states stabilized with nucleotide analogues or trapped during contraction by rapid freezing. The research focuses on the structure of the large waterbug Lethocerus sp. because its filament lattice is the best ordered of all known muscles thereby making it an excellent candidate for 3-D imaging as well as facilitating the trapping of many crossbridges into similar structures. Structures observed by 3-D electron microscopy of sectioned muscle will be correlated with X-ray diffraction of native muscle and mechanical measurements on the stabilized muscle or mechanical traces made prior to freezing. Our reconstruction work will continue to focus on thin 12-30 nm sections because these specimens yield the highest detail on crossbridge structure. Oblique section reconstruction will continue to be used to produce averaged images whose transforms are readily comparable with X-ray diffraction of the native muscle. Images of crossbridges in states with a relatively high variation in structure will be studied using plus/minus 70 degrees tilt series reconstructions that do not average crossbridges. We will then explore methods for aligning, classifying and averaging 3-D crossbridge structures. Finally, we will correlate variations in crossbridge structure observed in different states with the recent atomic models of actin and the myosin motor domain. These efforts will being with modelling the known structures of myosin and actin into 3-D reconstructions of rigor and muscle treated with the non-hydrolyzable analog AMPPNP.